Cells must maintain the integrity of their genomes in order to propagate and survive. However, genomes are constantly challenged by damage from endogenous metabolites and environmental sources that pose impediments to replication and transcription. Special risks arise during replication. If the replication fork is not protected, replisome stalling at lesions can ultimately lead to strand breakage, loss of genetic information, and genomic instability. To face these challenges, organisms evolved multiple DNA repair and checkpoint pathways that must be coordinated with each other, as well as with DNA replication. Breakdown either in any of these processes or their coordination can corrupt genome integrity and cause human disease phenotypes ranging from aging to cancer. This proposal aims to understand the biochemical basis and biological significance of two unexpected findings. First, the DNA repair protein XPG is up-regulated in S-phase and localizes to foci containing proteins that repair damaged replication forks. Second, XPG interacts physically and functionally with WRN, which is known to be important for maintaining genomic integrity during S-phase, and also interacts directly with the RAD51 recombinase. The central hypothesis to be tested is that XPG has novel roles in replication fork maintenance both cooperatively with WRN and through facilitation of RAD51-mediated homologous recombination. The proposed approaches harness the complementary biochemical and cell biological expertise of two established laboratories and their collaborators. Aim 1 will define the role of XPG at replication forks and characterize the biological consequences of its loss. Aim 2 will test the hypothesis that during S-phase XPG functions with WRN at a subset of stalled replication forks and determine the conditions under which they interact in cells. Aim 3 will investigate a proposed role for XPG as a mediator of homologous recombinational repair of replication-associated DNA double-strand breaks. The proposed studies will define the molecular basis for interactions among the DNA repair processes mediated by XPG and WRN and their novel role in preventing loss of genomic integrity during S phase, with implications both for informed regulation of environmental exposures and rational development of novel cancer therapies.